Certain pyridyl hydrazines and hydrazides useful for protein labeling

ABSTRACT

Bifunctional aromatic compounds which are capable of linking metal ions to biologically useful molecules. The bifunctional compounds are characterized as having a hydrazine or hydrazide group and a protein reactive group. The hydrazine or hydrazide group may be protected as a lower alkyl hydrazone. Conjugates of the bifunctional compounds with macromolecules are also described and labelled macromolecules comprised of the conjugates and metal ions are provided. Additionally, a method is provided for forming a labelled macromolecule by reacting a conjugate with a metal species The compounds and method of this invention are particularly useful in the fields of biology and medicine for imaging and/or therapy.

RELATED APPLICATION

This is a continuation of application Ser. No. 07/483,201 filed on Feb.21, 1990, which was abandoned upon filing hereof which is acontinuation-in-part of Ser. No. 07/315,270 filed Feb. 24, 1989,abandoned.

BACKGROUND OF THE INVENTION

This invention relates to bifunctional compounds capable of linkingmetal ions, particularly technetium and rhenium, to biologically usefulmolecules.

Because of their high biological specificity, certain macromolecules(e.g., monoclonal antibodies) have been used to target radioisotopes tospecific in vivo sites for the purpose of imaging and/or therapy. Theuse of the metastable isotope of technetium, ^(99m) Tc, in diagnosticnuclear medicine is well established and the beta-emitting isotopes ofrhenium ¹⁸⁶ Re, ¹⁸⁸ Re and ¹⁸⁹ Re can be used therapeutically. A numberof methods for attaching technetium to macromolecules have beendescribed. Some of these methods involve the reduction of disulfidegroups in the macromolecule (usually an immunoglobulin) to thiols andthe subsequent use of these groups to bind reduced Tc (e.g., McKenzie etal., International Publication #WO 87/04164; and Bremer et al., EPO 271806 A2). Methods of this type have several potential disadvantages. Thereduction of disulfide units can lead to protein de-naturation and asubsequent loss in biological specificity. Also, the method cannot beused to label macromolecules lacking disulfide moieties.

Alternatively, ^(99m) Tc can be linked to macromolecules viabifunctional chelates such as DTPA (D. Lanteigne and D. J. Hnatowich,Int. J. Appl. Radiat. Isot., Vol. 35(7), p. 617 (1984), chelatingthiosemicarbazones (Y. Arano et al., Int. J. Nucl. Med. Biol., Vol. 12,p. 425 (1985), and diamide-dithiol ligands (A. Fritzberg, EuropeanPatent Appl. EP 88256 2A). Problems associated with these methodsinclude significant nonspecific binding of technetium (binding to theprotein at sites other than the chelating group) and slow kinetics ofTc-labelling.

Accordingly, it is the object of the present invention to provide newbifunctional molecules having hydrazine or hydrazide groups and proteinreactive groups which can be used to link metal ions, such as ^(99m) Tc,to macromolecules.

Another object of the present invention is to provide a method forlabelling macromolecules with metal ions in which binding of the metalat sites other than the chelating group is minimal, and in whichlabelling occurs at a relatively fast rate (less than one hour at roomtemperature).

SUMMARY OF THE INVENTION

According to the invention, novel bifunctional hydrazine and hydrazidecompounds, as well as conjugates thereof, are provided. Methods oflabelling the conjugates with metal ions are also provided.

Broadly, the hydrazine and hydrazide compounds described herein arebifunctional aromatic hydrazines or hydrazides having a protein reactivesubstituent and a negative counterion. A modification of this inventionis also provided in which the hydrazine or hydrazide function isprotected as a lower alkyl hydrazone.

In another embodiment of the invention, conjugates are formed byreacting bifunctional hydrazine or hydrazide compounds of the inventionwith macromolecules such as proteins, polypeptides or glycoproteins. Thebifunctional compounds react with nucleophilic groups on themacromolecules (e.g. lysine residues) to yield conjugates containingfree hydrazine/hydrazide groups.

In a third embodiment, labelled macromolecules comprised of conjugatesand metal ions are formed.

In a fourth embodiment, a method is provided for labellingmacromolecules by reacting a conjugate of the invention with a metalspecies.

DETAILED DESCRIPTION OF THE INVENTION

The novel hydrazine and hydrazide compounds of the present invention arerepresented by one of the following formulas (I) or (II): ##STR1##wherein:

A is a carbon or nitrogen atom;

B is a carbon atom;

D is a direct bond (to the 2-, 3-, or 4-position of the ring), ##STR2##

E is C═O or together with forms a maleimidyl group;

F is a group readily replaced by a primary amine in neutral or basicaqueous media when E is C═O or together with E forms a maleimidyl group;

R is a hydrogen or a lower alkyl group;

R' and R" may be the same or different and are selected from hydrogenand lower alkyl; and

X is a negative counterion.

Another embodiment of the invention includes compounds of the formula(III): ##STR3## where R, R', R", E and F have the values given above.

When E is carbonyl C═O, F is any group which, in combination with theattached carbonyl group, forms an active ester or active amide. Examplesof suitable species for F include such diverse groups asN-oxysuccinimidyl, tetrafluorophenolate, N-oxybenztriazole andimidazolate. These examples are not intended to be construed as limitingthe scope of the invention.

Suitable groups for R, R' and R" include, but are not limited to, thefollowing: H, CH₃, C₂ H₅, and C₃ H₇.

Examples of useful of X ions are halides, nitrate, trifluoroacetate,tetrafluoroborate and sulfate. These examples are not intended to limitthe scope of suitable counterions.

The above-described compounds are stable, isolable derivatives ofmolecules that contain two cross-reactive moieties: ahydrazine/hydrazide group and a protein reactive group such as an activeester, active amide or maleimido group.

In the synthesis of these stable derivatives, an acid labile protectinggroup such as t-butoxycarbonyl (t-BOC) is removed from thehydrazine/hydrazide under anhydrous acidic conditions, leaving theprotein reactive group unchanged and the hydrazine/hydrazide group in anunreactive, protonated form. Alternatively, the hydrazine/hydrazidegroup can be protected as a lower alkyl hydrazone.

When a bifunctional compound having a protonated (or hydrazoneprotected) hydrazine/hydrazido function is then combined with amacromolecule such as a protein, polypeptide or glycoprotein in neutralor slightly basic media, preferably a pH of about 7-8.5, theprotein-reactive part of the compound will react with nucleophilicgroups on the protein, polypeptide or glycoprotein (e.g., amine groupssuch as lysine residues) to yield a conjugate containing freehydrazine/hydrazide groups. In the case of hydrazone conjugates, thefree hydrazine/hydrazide is formed by dialysis into an acidic (pH 5.6)buffer. Because this type of conjugate includes a hydrazine orhydrazide, a strong metal binding group, it will then readily react whenmixed with a suitable metal species in acidic media to yield a labelledprotein, polypeptide or glycoprotein.

The metal species may be, for example, a reduced Tc species formed byreacting TcO₄ ⁻ with a reducing agent, for example, stannous ion, in thepresence of a chelating oxygen ligand (e.g. glucoheptonate). Examples ofsuitable reduced Tc species include Tc-glucoheptonate, Tc-gluconate,Tc-2-hydroxyisobutyrate, Tc-lactate and Tc-4,5-dihydroxy 1,3-benzenedisulfonate. Other metals and ligands are also within the scope of theinvention.

A Tc labelling process can be conveniently performed in an aqueousbuffer, preferably at a pH of about 4.5-6.5, in one hour or less.Reaction with other suitable metal species occurs in a similar mannerunder similar conditions.

Radiochemical yield as determined by high performance liquidchromatography (HPLC) and thin layer chromatography (TLC) using Tc is≧90%. Treatment of protein with nonlinkable analogs, (i.e., compoundswithout a protein reactive carbonyl group, such as 4-hydrazinobenzoicacid or 6-hydrazinopyridine-3-carboxylic acid) does not yield proteincapable of significant Tc binding, thus demonstrating the highspecificity of this technique.

The technetium atoms are believed to be bound to the conjugate via ahydrazido or diazenido linkages: ##STR4## Wherein:

L is an ancillary dioxygen ligand.

Examples of this type of linkage have been described for Mo and Re(Comprehensive Coordination Chemistry, Vol. 2, G. Wilkinson, ed.,Pergamon (Oxford) 1987, p. 130-151) and several analogous complexes of⁹⁹ Tc have been prepared by the reaction of an organohydrazinederivative and a Tc(V) oxo species.

The above labelling scheme has been used to label polyclonal human IgGand the Fc region of human IgG. The Tc-conjugates have been used toimage focal sites of infection in a rat model. The labelling scheme hasalso been used to label fragment E₁ (see L. C. Knight et al, J. Clin.Invest., Vol. 72, 1983, p. 2007-2013) which was used to image thrombi ina rabbit model for deep vein thrombosis and the monoclonal antibody 5E8.

EXAMPLES

The NMR and IR data given in the examples was obtained as follows:

¹ H NMR spectra were recorded on an 80 MHz IBM AF-80 Spectrometer. All ¹H NMR results were recorded in DMSO-d₆ unless otherwise indicated. IRspectra were recorded on a Perkin-Elmer 598 infrared spectrometer. NMRand IR spectra were consistent with assigned structure.

Compound names given in brackets below the title compounds in thevarious examples conform to Chemical Abstracts service indexnomenclature. Reaction schemes are illustrated in the accompanying FIGS.1-8, respectively.

Scheme 1: corresponds to FIG. 1 involves Synthesis of Succinimidyl of4-Hydrazino Benzoate hydrochloride.

Scheme 2: corresponds to FIG. 2 involves Synthesis of Succinimidyl of6-Hydrazino Pyridine-3-carboxylate hydrochloride.

Scheme 3: corresponds to FIG. 3 involves Synthesis of Succinimidyl of4-Hydrazido terephthalate hydrochloride.

Scheme 4: corresponds to FIG. 4 involves Synthesis of 5-Maleimidyl2-Hydrazino pyridine hydrochloride.

Scheme 5: corresponds to FIG. 5 involves Synthesis of Succinimidyl2-(2-Propenyl Hydrazone)nicotinate

Scheme 6: corresponds to FIG. 6 involves Synthesis of Succinimidyl2-(2-(1-propenyl hydrazone))thiazole-4-carboxylate.

Scheme 7: corresponds to FIG. 7 involves Synthesis of Succinimidyl2-(2-methenyl hydrazone)thiazole-4-carboxylate

Scheme 8: corresponds to FIG. 8 involves Synthesis of Succinimidyl4-thiosemicarbazido benzoate hemihydrochloride.

EXAMPLE 1 Preparation of succinimidyl 4-hydrazinobenzoate hydrochloridehydrochloride [2.5-pyrrolidinedione,1-[(4-hydrazinobenzoyl)oxy]-monohydrochloride]

4-Hydrazinobenzoic acid,2-(t-butoxycarbonyloxyimino)-2-phenylacetonitrile (BOC-ON),dicyclohexylcarbodiimide and N-hydroxysuccinimide were purchased fromAldrich Chemicals (Milwaukee, Wis.).

Synthesis of 4-BOC-hydrazinobenzoic acid--To a stirred solution of4-hydrazinobenzoic acid (1 equivalent) and triethylamine (3 equivalents)in dimethylformamide (5 mg/1) was added dropwise a solution of BOC-ON (1equivalent) in dimethylformamide. The reaction mixture was stirred atroom temperature for 3 hours. Ten percent aqueous hydrochloric acid wasadded and subsequently the solution became cloudy. The solution wasextracted with ethyl acetate and the combined organic extracts werewashed with water, dried over magnesium sulfate, filtered andconcentrated under reduced pressure to give a brown solid. The solid wasrecrystallized from chloroform to give the desired product as a palebrown solid; yield 69%. Analysis:

Calculated for C₁₂ H₁₆ n₂ O₄ : C 57.13; H 6.39; N 11.10. Found: C 57.02;H 6.13; N 11.61.

¹ H NMR δ: 1.45(s,9H),6.77(d,J_(ab) =8.6 Hz,2H),7.85(d,J_(ab) =8.6Hz,2H)

Synthesis of succinimidyl 4-BOC-hydrazinobenzoate

[Hydrazinecarboxylic acid,2-[4-[[(2,5-dioxo-1-pyrrolidinyl)oxylcarbonyl] phenyl]-1,1-dimethylethylester]

To a solution of 4-BOC-hydrazinobenzoic acid (1 equivalent) andN-hydroxysuccinimide (1 equivalent) in dioxane (10 ml/g of acid) wasadded dropwise a solution of dicyclohexylcarbodiimide (1 equivalent) indioxane (5 ml/g). The reaction mixture was stirred at room temperaturefor 16 hours. Acetic acid (0.5 ml) was then added and stirring wascontinued for 1 hour. The reaction mixture was filtered to remove theurea byproduct. The filtrate was concentrated under reduced pressure togive a brown solid which was treated with ether and the solids wereisolated by filtration to give a pale brown solid; yield 86%.

Analysis: Calculated for C₁₆ H₁₉ N₃ O₆ : C 55.01; H 5.48; N 12.03;Found: C 55.17; H 5.84; N 11.86.

¹ H NMR δ: 1.47(s,9H),2.88(s,4H),6.85(d,J_(ab) =8.9 Hz,2H) 8.04(d,J_(ab)=8.9 Hz,2H)

Synthesis of succinimidyl 4-hydrazinobenzoate hydrochloride - To asolution of hydrogen chloride in dioxane (50 ml/g of ester; prepared bybubbling hydrogen chloride into dioxane for approximately five minutes)was added succinimidyl 4-BOC-hydrazinobenzoate (1 equivalent). Thereaction mixture was stirred at room temperature. The reaction mixturewas never homogeneous, however the color was initially pale brown andover 2 hours became orange. The reaction mixture was filtered and washedwith ether to give a pale yellow solid; yield 72%; m.p. 203.5°-205° C.

Analysis: Calculated for C₁₁ H₁₂ ClN₃ O₄ : C 46.25; H 14.23; Cl 12.40; N14.71; Found: C 46.74; H 4.38; Cl 12.24; N 14.26.

¹ H NMR δ: 2.87(s,4H),7.05(d,2H,J_(ab) =8.9 Hz)7.97(d,2H,J_(ab) =8.9 Hz)

EXAMPLE 2

Preparation of succinimidyl 6-hydrazinopyridine-3-carboxylatehydrochloride

[2,5-pyrrolidinedione, 1-[[(6-hydrazino-3-pyridinyl)carbonyl]oxy]-,monohydrochloride]

6-chloronicotinic acid, di-t-butyl dicarbonate and 85% hydrazine hydratewere purchased from Aldrich Chemicals (Milwaukee, Wis.).

Synthesis of 6-hydrazinopyridine-3-carboxylic acid

6-Chloronicotinic acid (8.0 g) was added to 85% hydrazine hydrate (35ml). The reaction mixture was placed in a 100° C. oil bath for 4 hours.The homogeneous reaction mixture was concentrated to dryness to give awhite solid. The solid was dissolved in water and on acidification to pH5.5 with concentrated hydrochloric acid a precipitate formed. Theprecipitate was isolated by filtration and the solid was washed with 95%ethanol and ether to give 6.0 g of a pale brown solid; yield 77% m.p.292°-293° C.;

Analysis: Calculated for C₆ H₇ N₃ O₂ ; C 47.06; H 4.61; N 27.44; Found:C 46.83; H 4.38; N 27.27.

¹ H NMR- δ: 6.69(d,J=8.8 Hz,1H),7.84(dd,J=2.4,8.8 Hz,1H), 8.51(d,J=2.4Hz,1H)

Synthesis of 6-BOC-hydrazinopyridine-3-carboxylic acid

To a solution of 6-hydrazinopyridine-3-carboxylic acid (1.4 g; 9.8mmol); triethylamine (1.2 ml; 11.8 mmol) in dimethylformamide (10 ml)was added di-t-butyldicarbonate (2.13 g; 9.8 mmol). The reaction mixturebecame homogeneous over 1 hour and stirring was continued for 16 hoursat room temperature. The reaction mixture was concentrated to drynessunder reduced pressure to give a brown solid. The residue was dissolvedin a minimum amount of ethyl acetate and filtered through silica gel 60(230-400 mesh) using ethyl acetate as eluent. The eluent wasconcentrated to dryness. The product was used without furtherpurification.

¹ H NMR δ:1.40(s,9H),6.52(d,J=8.8 Hz,1H)7.97(dd,J=2.4, 8.8Hz,1H),8.58(d,J=2.4 Hz,1H)

Synthesis of succinimidyl 6-BOC-hydrazinopyridine-3-carboxylate

Hydrazinecarboxylic acid,2-[5-[[(2,5-dioxo-1-pyrrolidinyl)oxylcarbonyl]-2-pyridinyl]-,1,1-dimethylethyl ester]

To a solution of 6-BOC-hydrazinopyridine-3-carboxylic acid (1.45 g; 5.75mmol) and N-hydroxysuccinimide (0.66 g; 5.75 mmol) in dimethylformamide(15 ml) was added a solution of dicyclohexylcarbodiimide (1.18 g; 5.75mmol) in dimethylformamide (5 ml). The reaction mixture became cloudyafter 1 hour and stirring was continued for 16 hours at roomtemperature. The reaction mixture was filtered and the filtrate wasconcentrated to dryness to give a brown solid residue. The residue wasdissolved in a minimum amount of ethyl acetate and filtered throughsilica gel 60 (230-400 mesh) using ethyl acetate as eluant. The eluantwas concentrated to dryness to give a pale yellow solid which wasrecrystallized from ethyl acetate/hexanes; yield 60%; m.p. 169.5°-172°C.;

Analysis: Calculated for C₁₅ H₁₈ N₄ O₆ ; C 51.43; H 5.18; N 15.99;Found: C 51.81; H 5.26; N 15.60.

¹ H NMR δ:1.41(s,9H),2.87(s,4H)6.64(d,J=8.8 Hz,1H)8.08 (dd,J=2.4,8.8Hz)8.73(d,J=2.4 Hz,1H)

Synthesis of succinimidyl 6-hydrazinopyridine-3-carboxylatehydrochloride--A solution of hydrogen chloride in dioxane was preparedby bubbling anhydrous hydrogen chloride into dioxane (20 ml) at amoderate rate for 10 min. Succinimidyl6-BOC-hydrazinopyridine-3-carboxylate (100 mg) was dissolved in dioxane(2 ml) and HCl/dioxane (2 ml) was added and the reaction mixture wasstirred at room temperature. After 5 minutes the solution became cloudyand a precipitate formed. Stirring was continued for 4 hours. The cloudyreaction mixture was filtered to give 55 mg of a white solid; yield 67%;

Analysis: Calculated for C₁₀ H₁₁ ClN₄ O₄ ; C 41.87; H 1- 3.87; Cl 12.37;N 19.53; Found: C 41.92; H 3.90; Cl 12.30; N 19.47.

¹ H NMR δ:2.88(s,4H),7.01(d,J=8.8 Hz,1H)8.19(dd,J=2.4, 8.8Hz,1H)8.83(d,J=2.4 Hz,1H)

EXAMPLE 3

Preparation of succinimidyl 4-hydraziodoterephthalate hydrochloride

[Benzoic acid, 4-[[(2,5-dioxo-1-pyrrolidinyl) oxylcarbonyl]-, hydrazide,monohydrochloride]

Mono-methyl terephthalate, oxalyl chloride, t-butyl carbazate,dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) werepurchased from Aldrich Chemicals (Milwaukee, Wis.).

Synthesis of methyl tereohthalate chloride--To a stirred solution ofmono-methyl terephthalate (1 equivalent), toluene (30 ml/gm of ester)and 3 drops of DMF was added dropwise oxalyl chloride (2.0 equivalents).The reaction mixture was stirred at 45° C. for 16 hours. The solutionwas concentrated under reduced pressure to give the desired product as apale yellow solid. The product was used without further purification;yield 82.0%; m.p. 50°-52° C. IR (thin film); 2970, 1775, 1720, 1430,1400, 1280, 1105, 880 cm⁻¹.

¹ H NMR δ:3.97(s,3H),8.14(s,4H).

Synthesis of methyl 4-BOC-hydrazidotereohthalate [4-benzenedicarboxylicacid, monomethyl ester, 2-[(1,1-dimethylethoxy) carbonyllhydrazide]--Toa vigorously stirred mixture of t-butyl carbazate (1 equivalent),methylene chloride (20 ml/gm) and 25% sodium bicarbonate (2.0equivalents) was added dropwise a solution of methyl terephthalatechloride (1 equivalent) in methylene chloride (40 ml/gm). The reactionmixture was stirred at 20° for 1/2 hour. The phases were separated andthe aqueous phase was extracted with methylene chloride. The combinedorganic phases were washed with 10% hydrochloric acid and brine. Theorganic phase dried (MgSO₄), filtered and concentrated to give a whitesolid; yield 91.7%; m.p. 197°-199°. IR (KBr): 3010, 1720, 1670, 1430,1270, 1220, 1130, 1100, 1030, 870, 750 cm⁻¹.

¹ H NMR(CDCl₃):δ1.49(s,9H),3.93(s,3H),7.83 (J_(AB) =8 Hz,2H),8.07(d,J_(AB) =8 Hz,2H).

Synthesis of 4-BOC-hydrazidotereohthalic acid--To a solution of methyl4-BOC-hydrazidoterephthalate (1 equivalent) in methanol (50 ml/gm) wasadded sodium hydroxide (10.0 equivalents). The reaction was stirred atroom temperature for 16 hours. The reaction mixture was concentratedunder reduced pressure to remove the methanol. Water was added and thesolution was carefully acidified to pH 1.0. The acidic solution wasextracted with ethyl-acetate and the organic extract was dried (MgSO₄),filtered and concentrated to dryness under reduced pressure to give awhite solid; yield 87.5%; m.p. 208°-210°.

¹ H NMR δ:1.41 (s,9H),7.97(d,J=2.4 Hz,4H),8.90(m,1H) 10.3(m,1H).

Synthesis of succinimidyl 4 BOC-hydrazidotereohthalate[Hydrazinecarboxylic acid,2-[4-[[(2,5-dioxo-1-pyrrolidinyl)oxylcarbonyl]benzoyl]-,1,1-dimethylethyl ester]--To a solution of 4-BOC-hydrazidoterephthalicacid (1 equivalent) and N-hydroxysuccinimide (1 equivalent) in DMF (10ml/gm) was added dropwise a solution of DCC (1 equivalent) in DMF (5ml/gm). The reaction mixture was stirred at 20° for 16 hours. Aceticacid (0.5 ml) was then added and stirring was continued for 1 hour. Thereaction mixture was filtered to remove the urea byproduct. The filtratewas concentrated under reduced pressure to give a yellow brown oil.Flash vacuum chromatography (hexanes/ethyl acetate (7/3)) was used toisolate the product; yield 47.8%, m.p. 182°-185°. IR (KBr): 3330, 3230,2990, 1770, 1740, 1660, 1530, 1500, 1370, 1280, 1200, 1150, 1070, 1000,870, 790, 640 cm⁻¹.

¹ H NMR (CDCl₃)δ:1.50(s,9H),2.91(s,4H),6.70(m,1H),7.91(d,J_(AB) =8.8Hz,2H), 8.20(d,J_(AB) =8.8 Hz,2H).

Analysis: Calculated for C₁₇ H₁₉ N₃ O₇ : C 54.11; H 5.07; N 11.13;Found: C 53.66; H 5.15; N 11.09.

Synthesis of succinimidyl-4-hydrazidotereohthalate hydrochloride--To asolution of hydrogen chloride in tetrahydrofuran (50 ml/gm; prepared bybubbling hydrogen chloride into tetrahydrofuran for approximately tenminutes) was added succinimidyl 4-BOC-hydrazidoterephthalate (1equivalent). The reaction mixture was homogeneous for 1 hour; then over4 hours a pale white precipitate formed. The reaction mixture wasfiltered and washed with ether to give the desired product as a palewhite solid; yield 37.7%; m.p. 278°-280°. IR(KBr): 3400, 3200, 2800,2600, 1770, 1730, 1690, 1530, 1490, 1290, 1240, 1070, 1000, 870, 730,640, 610 cm⁻¹.

¹ H NMR δ:2.91 (s,4H),8.11(d,J_(AB) =8.8 Hz, 2H),8.25(d,J_(AB) =8.8Hz,2H). Analysis: Calculated for C₁₂ H₁₂ ClN₃ O₅ : C 45.95; H 3.86; Cl11.30; N 13.40; Found: C 45.84; H 3.91; Cl 11.37; N 13.33.

EXAMPLE 4

Preparation of 5-maleimidyl-2-hydrazinopyridine hydrochloride[1H-pyrrole-5-dione, 1-(6-hydrazino-3-pyridinyl)-, monohydrochloride].

2-chloro-5-nitropyridine, hydrazine hydrate, di-tert-butyl dicarbonate,10% palladium on charcoal, maleic anhydride, cobalt acetate and aceticanhydride were purchased from Aldrich Chemicals (Milwaukee, Wis.).

Synthesis of 2-hydrazino-5-nitropyridine--To a stirred solution ofhydrazine hydrate (30.0 equivalents), water (4 ml/gm of pyridine), andethanol (2 ml/gm of pyridine) was added 2-chloro-5-nitropyridine (1equivalent). The reaction mixture was stirred at 20° for 16 hours (avery thick green slurry forms). The precipitate was isolated byfiltration and the solid was washed with methanol and then ether to givea green solid. The product was used without further purification; yield77.3%; m.p. 205°-207°. IR (KBr): 3340, 3200, 2980, 1670, 1605, 1580,1485, 1420, 1330, 1300, 1120, 980, 830, 770 cm⁻¹.

¹ H NMR δ:4.64(br s,2H),6.76(d,J=8.8 Hz,1H),8.15(dd,J=2.4,8.8 Hz,1H),8.86(d,J=2.4 Hz,1H),9.12,(m,1H). Analysis: Calculated for C₅ H₆ N₄ O₂ :C 39.21; H 3.93; N 36.51; Found: C 38 96; H 3.92; N 36.35.

Synthesis of 2-(BOC-hydrazino)-5-nitropyridine--To a stirred solution of2-hydrazino-5-nitropyridine (1 equivalent), DMF (15 ml/gm of pyridine),and triethylamine (1.1 equivalents) was added dropwise a solution ofdi-tert-butyl dicarbonate (1.0 equivalent) in DMF (4 ml/gm ofdicarbonate). The reaction mixture was stirred at 20° for 48 hours. Thereaction mixture was concentrated under reduced pressure to a yellowbrown oil. Flash vacuum chromatography (hexanes/ethyl acetate (8/2)) wasused to isolate the product. The product was recrystallized from ethylacetate/hexanes; yield 63.4%; m.p. 135°-137°. IR (KBr): 3280, 2980,1710, 1600, 1500 1330, 1290, 1270, 1250, 1150, 1120, 1010, 830, 760,650, 500 cm⁻¹.

¹ H NMR δ: 1.41(s,9H),6.60(d,J_(AB) =8.8 Hz,1H),8.28(dd,J=2.4,8.8Hz,1H), 8.93(d,J_(AB) =2.4 Hz,1H),9.14(m,1H),9.56(m,1H). Analysis:Calculated for C₁₀ H₁₄ N₄ O₄ : C 47.24; H 5.55; N 22.03; Found: C 46.99;H 5.50; N 21.93.

Synthesis of 2-(BOC-hydrazino)-5-amino-pyridine--Into a Parrhydrogenation bottle was added 2-BOC-hydrazino-5-nitropyridine (1equivalent), 10% palladium on charcoal (0.3 gm Pd/gm of pyridine) andethanol (100 ml/gm of pyridine). The reaction was hydrogenated at 50 psiH2 for 2 hours at room temperature on a Parr hydrogenator. The reactionmixture was filtered through a filter cell plug and rinsed with ethanol.The yellow green solution was concentrated under reduced pressure togive a pale yellow solid. The product was recrystallized from ethanol;yield 81.64%; m.p. 140°-142°. IR (KBr): 3360, 3200, 2980, 1650, 1635,1580, 1490, 1390, 1360, 1300, 1260, 1160, 1020, 880, 860, 830, 750, 590,530 cm⁻¹.

¹ H NMR δ: 1.37(s,9H),6.34(d,J=8.8 Hz,1H),6.89(dd,J=2.4,8.8 Hz,1H),7.14(m,1H),7.49(d,J=2.4 Hz,1H),8.50(m,1H). Analysis: Calculated for C₁₀H₁₆ N₄ O₂ : C 53.55; H 7.19; N 24.98; Found: C 53.73; H 7.21; N 25.05:

Synthesis of 2-BOC-hydrazino-5-maleimidylpyridine [Hydrazine-carboxylicacid, 2-[5-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-2-pyridinyl]-,1,1-dimethylethyl ester]--To a stirred solution of 2-BOChydrazino-5-amino pyridine (1 equivalent) in acetone (50 ml/gm) wasadded maleic anhydride (1.1 equivalents). The reaction mixture wasstirred at 25° for 2 hours. To the reaction mixture was added aceticanhydride (1.2 equivalents), cobalt acetate (0.007 equivalents) andtriethylamine (0.3 equivalents). The reaction mixture was stirred at 60°for 2 hours. The color of the reaction began as bright yellow and endedas dark yellow. The reaction mixture was concentrated under reducedpressure to give a yellow brown oil. Flash vacuum chromatography(hexanes/ethyl acetate (8/2)) was used to isolate the product. Theproduct was recrystallized in ether/hexane; yield 28.3%; m.p. 182°-184°.IR (KBr): 3400, 3300, 3100, 2990, 1700, 1610, 1500, 1410, 1360, 1320,1270, 1210, 1150, 1050, 830, 750, 690 cm⁻ 1.

¹ H NMR δ:1.39(s,9H),6.58(d,J=8.8 Hz,1H),7.14(s,2H),7.45 (dd,J=2.4,8.8Hz,1H),7.94(d,J=2.4 Hz,1H),8.37(m,1H). Analysis: Calculated for C₁₄ H₁₆N₄ O₄ : C 55.26; H 5.30; N 18.41; Found: C 55.14; H 5.30; N 18.33.

Synthesis of 5-maleimidyl-2-hydrazinopyridine hydrochloride--A solutionof hydrogen chloride in dioxane was prepared by bubbling anhydroushydrogen chloride into dioxane (50 ml) at a moderate rate for 10minutes. 2-(BOC-hydrazino)-5-maleimidylpyridine (200 mg) was dissolvedin dioxane (5 ml) and HCl/dioxane (10 ml) was added and the reactionmixture was stirred at room temperature. After 30 minutes the solutionbecame cloudy and a precipitate formed. The reaction mixture was stirredat 25° for a total of 5 hours. The slurry was filtered and washed withether to give 50 mg of a white solid; yield 31.6%; m.p. 280°-290°(decomp.; yellow to brown). IR (KBr): 3440, 3100, 2580, 1720, 1610,1560, 1480, 1390, 1200, 1150, 830, 690 cm⁻¹.

¹ H NMR δ:7.0(d,J=8.8 Hz,1H),7.19(s,2H),7.63(dd,J=2.4,8.8 Hz,1H),8.15(d,J=2.4 Hz,1H). Mass spectrum: m/z=204 (M-HCL)⁺.

EXAMPLE 5 Preparation of Succinimidyl 2-(2-propenylhydrazone)nicotinate[Propanal,[5-[[(2,5-dioxo-1-pyrrolidinyl)oxylcarbonyl]-2-pyridinyl]hydrazone].

6-Hydrazinonicotinic acid was prepared as previously described, andpropionaldehyde was purchased from Aldrich Chemicals (Milwaukee, Wis.).

Synthesis of Succinimidyl 2-(2-propenylhydrazone)nicotinate--To asuspension of 6-hydrazinonicotinic acid (1 equivalent) in DMF (40 ml/g)was added propionaldehyde (3 equivalents). The reaction mixture wasstirred at ambient temperature for 1 hour. If the reaction mixture didnot become homogeneous the flask was gently heated with a heat gun untilthe reaction mixture became homogeneous. The reaction mixture was cooledto ambient temperature and a solution of N-hydroxysuccinimide (1equivalent) in DMF was added. Subsequently a solution of DCC (1equivalent) in DMF was added dropwise. The reaction mixture was stirredfor 16 hours at ambient temperature. The precipitate which formed wasremoved by suction filtration and the mother liquors were concentratedto dryness. The brown solid residue was suspended in ethyl acetate andstirred for 1 hour and filtered. A pale brown solid precipitated fromthe ethyl acetate solution and was isolated by filtration to give thedesired product; yield 65%.

^(H) NMR δ:1.06(t,3H),2.34(m,2H),2.86(s,4H),7.11(d,J=9.4 Hz,1H),7.54(t,1H,J=4.9 Hz),8.10(dd,J=9.44,2.33 Hz,1H),8.72(d,J=2.33 Hz,1H)Analysis: Calculated for C₁₃ H₁₄ N₄ O₄ : C 53.79; H 4.86; N 19.30;Found: C 53.66; H 4.89; N 19.12.

EXAMPLE 6 Preparation of Succinimidyl2-(2-(1-propenyl)hydrazonel)-thiazole-4-carboxylate [Propanal,[4-[[(2,5-dioxo-1-pyrrolidinyl)oxylcarbonyl]-2-thiazolyl]hydrazonel

Thiosemicarbazide and bromolactic acid were purchased from AldrichChemicals (Milwaukee, Wis.).

Synthesis of Propionaldehyde Thiosemicarbazone--To a solution ofthiosemicarbazide (1 equivalent) and propionaldehyde (1.5 equivalents)in MeOH was added a few drops of glacial acetic acid. The mixture washeated to reflux for 45 minutes. The reaction mixture was concentratedon the rotovap which caused a precipitate to form. The solids wereisolated by filtration, washed with ether and dried in vacuo: yield 71%

Synthesis of 2-(2-(1-propenylhydrazonel)) thiazole-4-carboxylic acidhydrobromide--To a solution of propionaldehyde thiosemicarbazone (1equivalent) in MeOH was added bromolactic acid (1 equivalent). Thereaction mixture was heated at reflux for 1 hour, then cooled to roomtemperature and the solvent was removed on the rotovap. The resultingyellow solid was triturated with MeOH/ether and a pale yellow solid wasisolated and washed with ether and dried in vacuo.

¹ H NMR δ:1.00(t,J=7.9 Hz,3H),2.15(m,2H),7.44(t,J=4.9 Hz).

Synthesis of Succinimidyl2-(2-(1-propenyl)hydrazonel-thiazole-4-carboxylate--To a solution ofacid (1 equivalent), N-hydroxysuccinimide (1 equivalent) andtriethylamine (1.5 equivalents) in DMF was added dropwise a solution ofDCC (1 equivalent) in DMF. The reaction mixture was stirred for 16 hoursat room temperature. The precipitate which formed was removed byfiltration and the mother liquors were concentrated to dryness. Ethylacetate was added to the residue and stirred for 1 hour. The insolubleswere removed by filtration and the mother liquor was concentrated todryness. The product was flash chromatographed using hexanes/ethylacetate (1/2) as eluant to give the desired product as an off-whitesolid; yield 35%. ¹ H NMR δ:1.08(t,J=7.9 Hz,3H),2.24(m,2H),7.40(t,J=4.9Hz,1H),8.11(s,1H).

EXAMPLE 7 Preparation of succinimidyl 2-(2-methylethenylhydrazone)thiazole-4-carboxylate

Synthesis of succinimidyl 2-(2-methylethenylhydrazone)thiazole-4-carboxylate -2-(methylethyenylhydrazone)-4-thiazolecarboxylic acid hydrobromide (1equivalent) (prepared according to the method of H. Johne, D. Seifert,S. Johne and E. Bulka, Pharmazie 33, 259 (1978)) was dissolved in DMF(20 ml/g). N-Hydroxysuccinimide (1 equivalent) and triethylamine (1.5equivalents) were added. To the homogeneous mixture a solution of DCC (1equivalent) was added dropwise over 15 minutes. The reaction mixture wasstirred for 16 hours at room temperature. The precipitate which formedwas removed by filtration and the mother liquor was concentrated todryness to give an orange-brown solid. The residue was suspended inethyl acetate and stirred at room temperature for 1 hour. Insolubleswere removed by filtration and the mother liquor was concentrated togive a brown solid. The solids were triturated with ether andre-isolated by filtration to give a yellow-brown solid: yield 40%. Asample of the product was filtered through a short plug of silica gelusing hexanes/ethyl acetate (2/1) as eluate. The eluant was concentratedto give the desired product as a pale yellow solid; m.p. 202°-205°(decomp.).

¹ H NMR δ:1.92(s,3H),1.96(s,3H),2.86(s,4H),8.11(s,1H).

Mass spectrum: m/z=297 (M+1)⁺

EXAMPLE 8 Preparation of Succinimidyl 4-thiosemicarbazidobenzoatehemihydrochloride [Hydrazinecarbothioamide,N-[4-[[(2,5-dioxo-1-pyrrolidinyl)oxyl]carbonyl]-phenyl]-,hemihydrochloride].

4-Aminocarboxylic acid was purchased from Aldrich Chemicals (Milwaukee,Wis.).

Synthesis of BOC-4-thiosemicarbazidobenzoic acid Hydrazinecarboxylicacid, 2-[[(4-carboxyphenyl)amino]thioxomethyl]-, 1-(1,1-dimethylethyl)ester]--To a solution of 4-isothiocyanatobenzoic acid (1 equivalent)(prepared according to the method of D. W. Browne and G. M. Dyson,J.Chem.Soc. 178 (1934)) and triethylamine (1.2 equivalents) in DMF wasadded to a solution of t-butyl carbazate (1 equivalent). The reactionmixture was stirred at room temperature for 3 hours and subsequentlyconcentrated to dryness. The residue was dissolved in ethyl acetate andwashed with 10% citric acid and brine. The organic phase was dried(MgSO₄), filtered and concentrated to give the desired product as anoff-white solid; yield 70%; m.p. 131°-133° (decomp.).

¹ H NMR δ:1.42(s,9H),7.67(d,J_(AB) =8.9 Hz,2H),7.87(d,J_(AB) =8.9Hz,2H).

Synthesis of Succinimidyl BOC-4-thiosemicarbazidobenzoate[Hydrazinecarboxylic acid,2-[[[4-[[(2,5-dioxo-1-pyrrolidinyl)oxyl]carbonyl]phenyl]amino]thioxomethyl]-1,1-dimethylethylester]--To a solution of acid (1 equivalent) and N-methylmorpholine (1.1equivalents) in acetonitrile was added a solution of succinimidyltetrachloroethyl carbonate (1 equivalent) in acetonitrile. The reactionmixture was stirred at room temperature for 16 hours. Ethyl acetate wasadded to the reaction mixture and the homogeneous solution was washedwith cold 5% citric acid, cold water, cold aqueous saturated sodiumbicarbonate solution, cold water and cold brine. The organic phase wasdried (MgSO₄), filtered and concentrated to give a pale yellow oil. Theoil was flash chromatographed on silica gel using hexanes/ethyl acetateas eluant. The product was isolated as a yellow oil which solidified onthe addition of ether. The solids were isolated by filtration to givethe desired product; yield 25%; m.p. 161°-163°.

¹ H NMR δ:1.52(s,9H),2.88(s,4H),7.74(d,J_(AB) =10.0 Hz,2H),8.05(d,J_(AB) =10.0 Hz,2H). Analysis: Calculated for C₁₇ H₂₀ N₄ SO₆ : C49.99; H 4.94; N 13.72 S 7.85; Found: C 50.05; H 4.95; N 13.64 s 7.95.

Synthesis of Succinimidyl 4-thiosemicarbazidobenzoatehemihydrochloride--To a suspension of BOC succinimidyl ester in etherwas added a solution of dry HCl(_(g)) in ether (prepared by bubbling HClgas into dry ether). The suspension was stirred at room temperature for16 hours. The reaction mixture was heterogeneous over the entire courseof the reaction. The solids were isolated by filtration to give thedesired hydrochloride salt product; yield 80%; m.p. 155°-160°.

¹ H NMR δ:2.88(s,4H), 8.01(s,4H). Analysis: Calculated for C₁₂ H₁₃ N₄.0.5 HCl: C 44.00; H 4.15; N 17.10 S 9.79; Found: C 44.56; H 3.88; N17.04 S 9.74.

EXAMPLE 9 Conjugation of IgG

To a solution of 10 mg IgG (MW =155,000) in 2 ml 0.1M sodium phosphatebuffer (pH 7.8) was added 17.2 μl of 30 mM succinimidyl4-hydrazinobenzoate hydrochloride in dimethylformamide. After stirringfor 5 hours at room temperature, the reaction mixture was dialyzedagainst 0.1M sodium acetate buffer (pH 5.2). The number of hydrazinogroups conjugated onto the protein was measured by the method of T. P.King et al. (Biochemistry, 25:5774, 1986). Briefly, thehydrazino-protein conjugate was reacted with 4-nitrobenzaldehyde toconvert the hydrazino groups into hydrazones. The number ofhydrazones/protein molecule were determined spectrophotometrically usingthe hydrazone of p-nitrophenylbenzaldehyde and phenylhydrazine (λ_(max)=412, ε=2.41×10⁴) as a standard. Modification yields of 25-35% wereobtained.

EXAMPLE 10 Labelling of Conjugated IgG with ^(99m) Tc

A DuPont Tc-glucoscan kit was reconstituted with 3 ml of watercontaining 10mCi of ^(99m) TcO₄ ⁻. 250 μl of this solution was mixedwith 250 μl of 1-5 mg/ml conjugated IgG in 0.1M sodium acetate buffer(pH 5.2). After incubation for 1 hour at room temperature >95% of theactivity was associated with the protein as determined by radiometricHPLC (TSK 3000 column) and instant thin layer chromatography (ITLC.)

800 uCi of Tc labelled IgG was injected into rats containing an abscessin one hind leg. At 24 hours the rats were sacrificed. The distributionof radioactivity was measured:

    ______________________________________                                                       % Injected                                                     Organ          dose/gram tissue                                               ______________________________________                                        Blood          1.36                                                           Kidney         1.11                                                           Infected Muscle                                                                              0.76                                                           Normal Muscle  0.12                                                           Liver          0.81                                                           ______________________________________                                    

Ratio Infected/Normal muscle=6.3

EXAMPLE 11 Conjugation of fragment E₁.

(DD)E protein was concentrated to between 5-10 mg/ml and modified with a20-fold molar excess of succinimidyl 6-hydrazinopyridine-3-carboxylatehydrochloride in 12.5 mM borate buffer at pH 8.5. After a 5 hourincubation (with gentle stirring) at 4° C., the sample was dialyzedapproximately 12 hours versus degassed nanopure water.

Fragment E₁ was separated from the modified DD(E) complex by making thesample 0.55M in acetic acid and diluting 1:1 V/V with 6M urea. The pH ofthe sample was adjusted to 5.5 with 10 N NaOH and the sample was thendialyzed against 10 mM citrate buffer (pH 5.7) to remove excessreagents. During dialysis, DD protein precipitates leaving modifiedfragment E₁ in solution. The DD precipitate was readily removed bycentrifugation.

Modified E₁ was labelled with Tc-99m via reaction with Tc-99mglucoheptonate, as previously described. The Tc-99m labelled E₁ was usedto image a thrombus in a rabbit model (see D. Collen et al., J. Clin.Invest. 71. p. 368-376 (1983)).

EXAMPLE 12 Conjugation of monoclonal antibody 5E8 (see E. A. Chen et al,Cancer Research, 49, p. 3642-3649 (1989)).

5E8 (at a concentration of 5-10 mg/ml) was modified with a 14-foldexcess of succinimidyl 6-hydrazinopyridine-3-carboxylate hydrochloridein 12.5 mM borate buffer at pH 8.5 (5 hours at room temperature). Theantibody conjugate was dialyzed against a 20 mM citrate buffer (pH 5.2100 mM in NaCl). After centrifugation to remove a small amount ofturbidity, the degree of modification (determinedspectrophotometrically, as previously described) was found to be 6.5hydrazine groups per protein molecule. Analysis by ELISA andimmunocytoadherence showed no loss in immunoreactivity.

The specific compounds and details of the method described above areexemplary and are not intended to limit the scope of the invention.

We claim:
 1. A hydrazine or hydrazide compound of the formula (I) or(II): ##STR5## wherein: A is a carbon or nitrogen atom;B is carbon ornitrogen atom except that only one of said terms A and B is nitrogen; Dis a direct bond or ##STR6## E represents ##STR7## and F is ##STR8## orE and F together represent ##STR9## r is hydrogen or lower alkyl; R' andR" independently represent hydrogen and lower alkyl; and X is a negativecounterion.
 2. The compound of the claim 1 wherein D is a direct bond tothe 4-position of the ring.
 3. The compound of claim 2 wherein E iscarbonyl and F is N-oxysuccinimidyl and X is selected from the groupconsisting of a halide, nitrate, trifluoroacetate, tetrafluoroborate andsulfate.
 4. The compound of claim 2 wherein R is hydrogen or methyl, Eis carbonyl, F is N-oxysuccinimidyl, and X is Cl.
 5. The compound ofclaim 4 wherein one of A and B is carbon and the other is nitrogen. 6.The compound of claim 4 wherein A is carbon, B is nitrogen, and R ishydrogen.